Toll-like receptors (TLRs) are type-I transmembrane proteins that recognize a variety of pathogen-associated molecular patterns (PAMPs) from bacteria, viruses and fungi. In this way PAMPs serve as a first-line of defense against invading pathogens. Human TLRs can elicit overlapping yet distinct biological responses due to differences in cellular expression and activation of downstream signal transduction pathways (Akira et al., Adv Immunol, 78: 1-56, 2001). TLRs are characterized by an ectodomain composed of leucine-rich repeats (LRRs) and a cytoplasmic domain, known as a Toll/interleukin-1 receptor (TIR) domain. The LRR-containing ectodomain is responsible for recognition of PAMPs, while the cytoplasmic domain is required for downstream signaling. Studies have shown that LRR8 is involved in DNA and RNA recognition, whereas LRR17 is involved in nucleic acid binding (Smits et al., Oncologist, 13: 859-875, 2008).
The TLRs located in the plasma membrane recognize bacterial membrane components, whereas the TLRs that detect nucleic acid-based ligands are predominately located within endosomal compartments. The nucleic acid-sensing TLRs include TLR3, TLR7, TLR8, and TLR9. TLR3 recognizes double-stranded RNA, TLR7 and TLR8 recognize single-stranded RNA, and TLR9 recognizes bacterial and viral DNA as well as synthetic oligodeoxynucleotides containing unmethylated CG dinucleotides (Akira and Hemmi, Immunol Lett, 85:85-95, 2003).
TLR8 belongs to the same subfamily as TLR7 and TLR9 and is highly homologous to TLR7 (Liu et al., Mol Immunol, 47:1083-90, 2010). Even so, the specificity of TLR8 for RNA and synthetic small molecules with a structure related to nucleic acids is not identical to that of TLR7 (Medzhitov et al., Immunol Rev, 173:-89-97, 2000). For instance, some ssRNA synthetic sequences containing repetitive A/U motifs are able to specifically activate TLR8 but not TLR7 (Gorden et al., J Immunol, 174:1259-68, 2005). Further, in humans, TLR8 is highly expressed in monocytes, macrophages, myeloid dendritic cells (mDC) and neutrophils, whereas TLR7 in blood cells is principally expressed in plasmacytoid dendritic cells (pDCs), B-cells, and neutrophils. Because of this difference in cellular expression, triggering by RNA through TLR7 in blood leads to a response dominated by Type I interferon (IFN) production, whereas activation through TLR8 induces multiple pro-inflammatory cytokines such as TNF, IL-12, IL-6, IL-8 and IL-1 (Barrat et al., J Exp Med, 202:1131-9, 2005; and Gorden et al., J Immunol, 174:1259-68, 2005).
TLRs have been implicated in various autoimmune and inflammatory diseases, with the clearest example being the role played by TLR9 and TLR7 in the pathogenesis of systemic lupus erythematosus (Barrat and Coffman, Immunol Rev, 223:271-283, 2008). Additionally, a TLR8 polymorphism has been associated with rheumatoid arthritis (Enevold et al., J Rheumatol, 37:905-10, 2010). Although various TLR7, TLR8 and TLR9 inhibitors have been described, additional TLR inhibitors are desirable. In particular, polynucleotides having inhibitory motifs for one or more of TLR7, TLR8 and TLR9 are needed to precisely inhibit an immune response in a subject (e.g., patient having an autoimmune disease or an inflammatory disorder).
Additionally, several polynucleotides have been identified, which inhibit R848-induced cytokine secretion by mouse splenocytes. Mouse TLR8, however, lacks the ability to respond to ssRNA ligands, RNA viruses or small molecules, all of which have been shown to activate human TLR8 (Heil et al., Science, 303:1526-9, 2004; Jurk et al. Nat Immunol, 3:499, 2002; Hemmi et al., Nat. Immunol, 3:196-200, 2002; and Lund et al., PNAS, 101:5598-603, 2004). Further, by comparing amino acid sequences, TLR8 of mice and rats was found to lack a five amino-acid sequence required for ligand recognition in man (Liu et al., Mol Immunol, 47:1083-90, 2010). Thus polynucleotides having inhibitory motifs for human TLR8 are desirable for use in human subjects.